Chondroitin sulphate (CS) is a complex natural polysaccharide belonging to the glycosaminoglycan (GAG) class, consisting of disaccharide sequences formed by residues of glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) sulphated in different positions and bonded by beta 1-3 bonds.
CS is present in animal tissues, with structural and physiological functions. Depending on its origin, CS mainly consists of variable percentages of two types of disaccharide unit monosulphated at position 4 or position 6 of GalNAc (disaccharides A and C respectively). However, disaccharides in which the sulphate groups are present in different numbers and different positions may be present in various percentages in the polysaccharide chains. The CS backbone also contains unsulphated disaccharide, generally in small quantities. Disulphated disaccharides having two sulphate groups bonded through the oxygen atom in various positions, such as position 2 of GlcA and 6 of GalNAc (disaccharide D), position 2 of GlcA and 4 of GalNac, or positions 4 and 6 of GalNAc (disaccharide E), can be present in the CS backbone in variable percentages, depending on the specific animal sources (Volpi N. J Pharm Pharmacol 61, 1271, 2009. Volpi N. J Pharm Sci 96, 3168, 2007. Volpi N. Curr Pharm Des 12, 639, 2006).
The repeating disaccharide unit found in CS has the following chemical formula:

wherein R2, R4 and R6 are independently H or SO3−.
The negative charges of the carboxylate and sulphate groups in the repeating disaccharide unit are neutralised by sodium ions.
The meanings of the acronyms most commonly used to identify the variously sulphated disaccharides are set out below:
Di-0S(R2 = H; R4 = H; R6 = H)Di-6S (C)(R2 = H; R4 = H; R6 = SO3−)Di-4S (A)(R2 = H; R4 = SO3−; R6 = H)Di-4,6diS (E)(R2 = H; R4 = SO3−; R6 = SO3−)Di-2,6diS (D)(R2 = SO3−; R4 = H; R6 = SO3−)Di-2,4diS (B)(R2 = SO3−; R4 = SO3−; R6 = H)Di-2,4,6triS(R2 = SO3−; R4 = SO3−; R6 = SO3−)
Samples of CS originating from different animal sources are also characterised by different molecular weights and charge densities, this latter parameter being directly correlated with the specific sulphated groups.
Table 1 shows the main disaccharides found in natural CS extracted from cartilage and other tissues of various animal species:
TABLE 1BovineChickenCSPorcine CSCSShark CSSkate CSSquid CSMn (kDa)12-17 9-14 8-1325-4027-3460-80Mw (kDa)20-2614-2016-2150-7050-70 80-120Polydispersity1.8-2.21.4-1.81.6-2.01.0-2.01.2-2.50.8-1.3indexDi-0S 6 6 83313Di-6S331420443915Di-4S618072324350Di-2,6diSNDNDND18130Di-4,6diSNDNDND2122Di-2,4diSNDNDND110Charge density0.90-0.960.92-0.960.90-0.941.15-1.251.08-1.201.00-1.20Ratio 4S/6S1.50-2.004.50-7.003.00-4.000.45-0.901.00-1.402.50-4.00Mn = number average molecular weight;Mw = weight average molecular weight;Polydispersity Index = Mw/Mn;Charge Density = the number of sulphate groups per disaccharide units;ND = Not identified.
As shown in Table 1, CS derived from land animals has similar molecular mass parameters (Mn and Mw), whereas it is different from that originating from fish species, which have higher molecular mass values. The terrestrial CS samples are also characterised by charge density (CD) values below 1.0, whereas the marine CS samples always have CD values exceeding 1.0. This characteristic is due to the different distribution of the sulphated disaccharides. Generally, disulphated disaccharides are found in trace amounts in terrestrial CS, and no polysulphated disaccharides (tri- and tetra-sulphates) are observed in natural CS.
The absence of tri- and tetra-sulphated disaccharides can easily be evidenced by analysis following digestion of the polysaccharide with chondroitinase ABC, a lytic enzyme specific for monosulphated disaccharides (Di-4S and Di-6S) and for unsulphated disaccharides (Di-0S), which are able to digest disulphated disaccharides but unable to hydrolyse the polysaccharide chain in correspondence with the polysulphated disaccharides. FACE (Fluorophore-Assisted Carbohydrate Electrophoresis) analysis of natural CS digested with chondroitinase ABC does not detect the electrophoresis bands characteristic of the partly undigested oligosaccharides which are found in synthetic or semisynthetic CS deriving from the prior art.
It is also well known that, due to biosynthesis processes, all natural CSs always show the simultaneous presence of monosulphated disaccharides at position 4 or 6 of GalNAc on the same polysaccharide chains (D'Arcy, S. M. et al., Carbohydr. Res., Vol. 255, Mar. 4, 1994, pp. 41-59; Hardingham, T. E. et al., Carbohydr. Res., Vol. 255, Mar. 4, 1994, pp. 241-54; Cheng, F. et al., Glycobiology, Vol. 2, No. 6, December 1992, pp. 553-61; Chai, W. et al., Anal. Biochem., Vol. 237, No. 1, May 15, 1996, pp. 88-102; Zaia, J. et al., Anal Chem., Vol. 73, No. 24, Dec. 15, 2001, pp. 6030-39; Desaire, H. et al., Anal Chem., Vol. 73, No. 15, Aug. 1, 2001, pp. 3513-20).
Different activities have been reported for CS in relation to its molecular structure (Kimata, K. et al., Mol. Cell. Biochem., Vol. 1, 1963, p. 211; Volpi, N., Biomaterials, Vol. 23, 2002, p. 3015; Volpi, N. and Tarugi, P., Biochimie, Vol. 81, 1999, p. 955; Volpi, N., Biomaterials, Vol. 20, 1999, p. 1359; Suzuki, S. et al., J. Biol. Chem., Vol. 243, 1968, p. 7).
CS has anti-inflammatory activity, and is currently recommended in the treatment of osteoarthritis (OA) as a Symptomatic Slow-Acting Drug for OsteoArthritis (SYSADOA) in Europe, in particular for the treatment of osteoarthritis of the knee (Jordan, K. M. et al., Ann. Rheum. Dis., Vol. 62, 2003, p. 1145), hip (Jordan, K. M. et al., Ann. Rheum. Dis., Vol. 62, 2003, p. 1145) and hand (Zhang, W. et al., Ann. Rheum. Dis., Vol. 66, 2007, p. 377) on the basis of clinical evidence and corresponding meta-analyses of numerous clinical trials. CS is also widely used as a nutraceutical in Europe and the USA, either alone or in combination with other ingredients (McAlindon, T. E. et al., JAMA, Vol. 283, 2000, p. 1469; Volpi, N. et al., Food Anal. Meth., Vol. 1, 2008, p. 195; Volpi, N. et al., Separation Sc., Vol 1, 2009, p. 22).
Commercial CS is obtained by extraction from animal tissue, such as bovine and porcine tissue (Fuentes, E. P. et al., Acta Farm. Bonaerense, Vol. 17, 1998, p. 135), bird tissue (Luo, X. M. et al., Poult. Sci., Vol. 81, 2002, pp. 1086-89) and fish cartilage (Sugahara, K. et al., Eur. J. Biochem., Vol. 239, 1996, p. 871; Lignot, B. et al., J. Biotechnol., Vol. 103, 2003, p. 281).
The animal origin of commercial CS involves safety problems associated with transmissible infectious agents that cause diseases such as bovine spongiform encephalopathy (BSE), and restricts the possible sources available to meet the growing worldwide demand. These factors have stimulated research into alternative methods of producing CS.
Intensive efforts have been made to find a biotechnological method of producing CS, using a micro-organism as source of a precursor polysaccharide which has a structure partly similar to that of CS and conducting chemical sulphation to produce a CS similar to the natural one.
One example of this strategy is the production of biotechnological CS from capsular polysaccharide K4 of E. coli O5:K4:H4, as described in EP 1304338 B1. Said patent discloses a process wherein polysaccharide K4 produced in liquid cultures is extracted and purified, and then redissolved and subjected to acid hydrolysis to eliminate the fructose residues bonded to the GlcA residues of the polymer. The defructosylated polymer, identical to the unsulphated backbone of CS(CH), is then sulphated at position 4 or position 6 of the GalNAc residue according to two different chemical synthesis methods. Said patent also discloses a third method whereby a disulphated CS in both positions 4 and 6 is obtained. The CS described therein has a content of at least 70% of sulphated polysaccharides consisting of mono- and/or di-sulphated at position 4 and 6 of the GalNAc residue, position 2′ of the GlcA residue being unsulphated, and has a molecular weight (Mw) of 6-25 kDa and a charge density (CD) of 0.7-2.0.
In EP 1304338 B1 the authors disclose and claim, depending on the synthesis strategy used, the possibility of:                (a) synthesising CS 4S by selectively protecting position 6 of all the N-acetylgalactosamine (GalNAc) residues present, thus obtaining a polymer selectively sulphated only at position 4 of all the N-acetylgalactosamine (GalNAc) residues; and        (b) obtaining a polymer in which, similarly, the hydroxyl groups at position 6 of all the GalNAc residues are sulphated, suitably protecting the hydroxyl residues present at position 4.        
In the process described in EP 1304338 B1, simultaneous sulphation therefore never takes place at positions 4 or 6 in the same chain, unlike the situation with natural CS.